Modification by ubiquitin has been implicated in the turnover of many proteins, such as cyclins, p53, and various oncoproteins. This grant focuses on the role of ATPases in the degradation of ubiquitin-conjugates. A family of 5 related but functionally distinct ATPases appears to reside within a multisubunit complex known as the 26S protease, which is essential for degradation of ubiquitin-conjugates. The importance of these ATPases is suggested by the strict ATP-dependence of ubiquitin-conjugate degradation, the requirement for all 5 genes for viability in yeast, and effects of point mutations in these genes on proteolysis. However, nothing is known about the mechanism by which these ATPases participate in protein degradation. We propose a molecular chaperone model for 26S ATPase function, namely that the these proteins use ATP hydrolysis to drive cycles of association and dissociation of the proteolytic substrate. We further propose that functional distinctions among 26S ATPases are at least partly attributable to their having distinct substrate binding specificities. The multiplicity of ATPases in the 26S protease, and their extreme evolutionary conservation, suggests that the role of ATP in 26S function is of fundamental importance but also poses difficulties in addressing these roles, since adding ATP to the complex may alter the functioning of not one but many of its components. In this proposal, we describe the use of yeast genetics to overcome this problem and to dissect systematically the role of ATP in proteolysis. We have constructed synoptic sets of mutations in the predicted ATP-binding regions of the 5 ATPase genes. Surprisingly, the mutations are for the most part nonlethal, although they result in pleiotropic phenotypes and dramatic protein stabilization effects. Using these mutations, the detailed in vivo functions of each ATPase activity can be determined. In particular, we will determine the role of each ATPase in the degradation of a variety of available in vivo substrates. We have developed a purification of the yeast 26S protease, which will allow us to study the ATPase mutants in vitro as well. Biochemical experiments will address specific mechanistic models for the individual role of each ATPase in the proteolytic process. For example, our hypothesis that these ATPases bind proteolytic substrates in an ATP- dependent manner will be examined by searching for peptide ligands, which may be used to study Yam-substrate interactions and their possible role in proteolysis. Finally, each ATPase will be localized within the architecture of the 26S protease by high-resolution immunoelectron microscopy. In summary, the powerful combination of biochemistry and genetics proposed below will provide insights into a novel ATPase particle, and allow us to test the molecular chaperone model of 26S ATPase function.